Platelet activation and aggregation, while needed for normal physiological functions such as hemostasis, can lead to a myriad of oft-lethal and highly debilitating conditions and pathologies when their regulatory mechanisms malfunction. These pathological conditions can be acute or chronic, and include acute coronary syndrome, myocardial infarction, unstable angina, stroke, coronary thrombosis, venous thrombosis, atherothrombosis, restenosis and so on. In the United States, Europe, and other industrialized nations, myocardial infarction due to rupture of atherosclerotic plaques is a leading contributor to morbidity and mortality. Acute plaque rupture exposes subendothelial collagen which promotes platelet activation and formation of a potentially occlusive thrombus at the site of vascular damage (Glass and Witztum, 2001; Rugged, 2002). Following their initial tethering to subendothelial collagen and matrix proteins, activation of transiently adhered platelets by autocrine mediators is critical for the propagation of the formative platelet thrombus. Reinforcement of the transient adhesive contacts by activating G protein-dependent shape change, granule release, and integrins permits growth of a stable thrombus that is resistant to the high shear stress of arterial blood flow (Jackson et al., 2003; Moers et al., 2003). Drugs that target the secondary autocrine mediators of platelet thrombus formation such as aspirin and thienopyridines have proven to be beneficial, however, many patients taking these drugs still sustain thrombotic events, and, therefore, might benefit from new therapeutics that interfere with matrix-dependent platelet activation (Bhatt and Topol, 2003).
Two distinct pathways act in parallel to activate platelets during hemostasis (Furie and Furie, 2008). As the blood vessel wall gets breached, platelets circulating in blood first encounter collagen embedded in the subendothelial matrix. As a first line of defense, exposed collagen initiates the accumulation and activation of platelets and starts the formation of a thrombus. As blood flows out further, it encounters a second line of defense, the tissue factor located in the medial and adventitial layers of the vessel wall, and a second independent pathway is triggered that also activates platelets to adhere to each other and form part of the developing thrombus. The tissue factor-initiated pathway generates thrombin which in turn cleaves protease-activated receptor 1(PAR1) on the human platelet surface, causing them to release adenosine diphosphate (ADP), serotonin, and thromboxane A2. In turn, these agonists recruit and activate other platelets, amplifying the signal in order to block off the breach in the vessel wall. The present invention, however, is based on discoveries that center around the other, collagen-initiated platelet activation pathway, i.e., the first line of defense in a thrombotic event.
Matrix metalloproteases (MMPs) have recently emerged as important mediators of platelet function and vascular biology. Initially described as extracellular matrix remodeling enzymes involved in tissue repair and cancer invasion (Egeblad and Werb, 2002), a renewed focus has centered on MMPs and the related metalloprotease disintegrins because of their prominence in vascular wall inflammation (Dollery and Libby. 2006) and thrombotic thrombocytopenic purpura (Levy et al., 2001). Endogenous platelet metalloproteases have been shown to damage platelet function by cleaving cell surface receptors and broad-spectrum metalloprotease inhibitors improve post-transfusion recovery of platelet concentrates (Bergmeier et al., 2003; Bergmeier et al., 2004; Stephens et al., 2004). Platelets express several metalloproteases including MMP-1, MMP-2, MMP-3, and MMP-14 on their surface (Chesney et al., 1974; Gait et al., 2002; Kazes at al., 2000; Sawicki et al., 1997). Notably, endogenous MMP-1 and MMP-2 can actually promote platelet aggregation but the cell surface target(s) and mechanism of activation have not been elucidated (Gait et al., 2002; Sawicki et al., 1997). A recent study that examined the effects of MMP-1 promoter polymorphisms in 2000 patients, found a significantly increased risk of myocardial infarction in patients with high promoter activity haplotypes and a significantly decreased risk in patients with low promoter activity haplotypes (Pearce et al., 2005).
It was recently shown that the G protein-coupled receptor, PAR1, is directly cleaved and activated on the surface of cancer cells by fibroblast-derived MMP-1 (Boire et al., 2005). PAR1 is the major thrombin receptor of human platelets (Coughlin, 2000; Leger et al., 2006b) and is an important mediator of platelet aggregation following tissue factor (TF)-dependent generation of thrombin (Mackman, 2004; Schwertz et al., 2006). However, under pathophysiologic conditions of acute plaque rupture, exposed collagen is the most efficient stimulus of the critical early events of platelet recruitment and propagation under arterial flow which could trigger metalloprotease activation on the platelet surface.